Promoter and vectors for plant transformation and methods of using same

ABSTRACT

The invention is directed to a promoter, designated MuB, sequences which hybridize to same and functional fragments thereof. The regulatory element of the invention provide improved expression in plants of operably linked nucleotide sequences. Expression vectors with the regulatory element is the subject of the invention, which may further include an operably linked nucleotide sequence. The invention is further directed to transformed plant tissue including the nucleotide sequence and to transformed plants and seeds thereof. The regulatory element is useful for driving gene or antisense expression or the like for the purpose of imparting agronomically useful traits such as, but not limited to, increase in yield, disease resistance, insect resistance, herbicide tolerance, drought tolerance and salt tolerance in plants.

BACKGROUND OF THE INVENTION

The expression of a heterologous nucleotide sequence in a plant cell isimpacted by regulatory nucleic acids. Promoters and terminators are twotypes of regulatory elements that impact expression of such operablylinked sequences. Promoters are vital molecular tools that have beenapplied widely in plant biotechnology to control the expression ofintroduced genes. A promoter is a nucleic acid sequence to which RNApolymerase must bind if it is to transcribe the linked gene intomessenger RNA and ultimately produce protein. A promoter may affect astructural gene operationally associated with the promoter in differentways. For example, it may enhance or repress expression of an associatedstructural gene, subject that gene to developmental regulation, orcontribute to the tissue-specific regulation of that gene. There aredifferent types of promoters used dependent upon the function desired.Constitutive promoters provide for expression throughout all tissues ofthe plant, where tissue preferred promoters will express at a higherrate in a (or a few) select tissue of the plant. Inducible promoters arethose which induce the regulatory affect of the promoter in response toa stimulus, which can be, for example, chemical, temperature, stress,wounding or other stimuli. The linked nucleotide sequence can performany of a wide variety of functions desired, whether it is repressing orinitiating expression of a trait or protein of interest, providing forover-expression, modifying metabolic and developmental pathways withinthe plant tissue, or the like.

Several promoters of plant and plant pathogen (bacterial and viral)origin have been used to direct transgene expression in plants.Prominent examples include the French bean beta-phaseolin promoter(Bustos et al., 1989), the mannopine synthase promoter of Agrobacteriumtumefaciens (Leung et al., 1991), and the 35S promoter of cauliflowermosaic virus (Guilley et al., 1982). These and several other promotersin widespread use in plants were originally developed and utilized indicot species. Despite the desire to identify constitutive promoterscapable of driving a relatively high level of gene expression in mosttissues of the plant, there remain few to choose from and there is anongoing need to identify promoters for use in expressing linkedsequences.

All references cited herein are incorporated herein by reference.

SUMMARY OF THE INVENTION

A promoter designated MuB has been identified, and function as apromoter demonstrated. The invention is further directed to sequenceswhich hybridize to same under highly stringent circumstances andfunctional fragments. In an embodiment, the regulatory element is usedto regulate high level, constitutive expression of linked nucleotidesequences.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the promoter sequence of the invention, SEQ ID NO: 1. theputative TATA box is underlined.

FIG. 2 shows the nucleotide sequence (SEQ ID NO: 1), including all 351base pairs of the MuB promoter, and the source of each homologoussequence from which a given segment is mostly comprised. The source andGenBank accession is as follows: A: MuA U.S. Pat. No. 6,222,096; B:AB000835; C: Y08502; D: U09376; E: AB005247; F: AF028711; G: AC002521;H: D12522; I: AB012243; J: AL022347.

FIG. 3 shows the homology between the MuB (SEQ ID NO: 1) and the CaMV35S promoter sequences (SEQ ID NO: 2) over a 351 bp overlap.

FIG. 4 shows the homology between the MuB (SEQ ID NO: 1) and the MuApromoter sequences (SEQ ID NO: 3) over a 352 bp overlap.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to a promoter, designated MuB,providing constitutive expression of an operably linked sequence. Theconstruction of this promoter provides a general method for thediscovery of novel sequences with utility as promoters. The presentinvention is also directed to DNA molecules including said promoter,such as a DNA construct comprising the promoter operably linked to oneor more genes or antisense DNA. The invention is further directed totransformed plant tissue including the DNA molecule and to transformedplants and seeds thereof. The promoter is useful for driving gene orantisense expression for the purpose of imparting agronomically usefultraits such as, but not limited to, increase in yield, diseaseresistance, insect resistance, herbicide tolerance, drought toleranceand salt tolerance in plants. Nucleotide sequences are described hereinthat regulate transcription with high constitutive expression in plantcells.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or an expression vector. This term alsoincludes those prokaryotic or eukaryotic cells that have beengenetically engineered to contain the cloned gene(s) in the chromosomeor genome of the host cell. The promoter is, in an embodiment,particularly useful for the expression of gene sequences in plants. Itcan be used in any plant species, including a dicotyledonous plant, suchas, by way of example but not limitation, tobacco, tomato, potato,soybean, cotton, canola, sunflower or alfalfa. Alternatively, the plantmay be a monocotyledonous plant, by way of example but not limitation,maize, wheat, rye, rice, oat, barley, turfgrass, sorghum, millet orsugarcane.

The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly other plants,or to synthesize synthetic sequences. In this manner, methods such aspolymerase chain reaction (PCR), hybridization, synthetic geneconstruction and the like can be used to identify or generate suchsequences based on their sequence homology to the sequences set forthherein. Sequences identified, isolated or constructed based on theirsequence identity to the whole of or any portion of the promotersequences set forth are encompassed by the present invention. synthesisof sequences suitably employed in the present invention can be effectedby means of mutally priming long oligonucleotides. See for example,Wosnick et al. (1987). In a PCR approach, oligonucleotide primers can bedesigned for use in PCR reactions to amplify corresponding DNA sequencesfrom cDNA or genomic DNA extracted from any plant of interest. Methodsfor designing PCR primers and PCR cloning are generally known in the artand are disclosed (Sambrook et al., 1989; Innis et al., 1990; Innis etal., 1995; Innis et al., 1999). Moreover, current techniques whichemploy the PCR reaction permit the synthesis of genes as large as 1.8kilobases in length. See Adang et al. (1993) and Bambot et al. (1993).Known methods of PCR include, but are not limited to, methods usingpaired primers, nested primers, degenerate primers, gene-specificprimers, vector-specific primers, partially-mismatched primers, and thelike. In addition, genes can readily be synthesized by conventionalautomated techniques.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the DNA sequences of theinvention. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed (Sambrook et al., 1989).

For example, the promoter sequence disclosed herein, or one or moreportions thereof, may be used as a probe capable of specificallyhybridizing to corresponding sequences. To achieve specifichybridization under a variety of conditions, such probes includesequences that are unique among the sequences to be screened and arepreferably at least about 10 nucleotides in length, and most preferablyat least about 20 nucleotides in length. Such sequences mayalternatively be used to amplify corresponding sequences from a chosenplant by PCR. This technique may be used to isolate sequences from adesired plant or as a diagnostic assay to determine the presence ofsequences in a plant. Hybridization techniques include hybridizationscreening of DNA libraries plated as either plaques or colonies(Sambrook et al., 1989).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na⁺ ion, typically about 0.01 to1.0 M Na⁺ ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1.0 M NaCl, 0.1% SDSat 37° C., and a wash in 0.1×SSC at 60 to 65° C.

In general, sequences that correspond to the nucleotide sequences of thepresent invention and hybridize to the nucleotide sequence disclosedherein will be at least 50% homologous, 70% homologous, and even 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%homologous or more with the disclosed sequence. That is, the sequencesimilarity between probe and target may range, sharing at least about50%, about 70%, and even about 85% or more sequence similarity.

Specificity is also the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation T_(m)=81.5° C.+16.6 (logM)+0.41(% GC)−0.61(% form.)−500/L,where M is the molarity of monovalent cations, % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % form. is the percentageof formamide in the hybridization solution, and L is the length of thehybrid in base pairs (Meinkoth and Wahl, 1984). The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of acomplementary target sequence hybridizes to a perfectly matched probe.T_(m) is reduced by about 1° C. for each 1% of mismatching; thus, T_(m),hybridization, and/or wash conditions can be adjusted for sequences ofthe desired identity to hybridize. For example, if sequences with 90%identity are sought, the T_(m) can be decreased 10° C. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (T_(m)); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (T_(m)); lowstringency conditions can utilize a hybridization and/or wash at 11 to20° C. lower than the thermal melting point (T_(m)). Using the equation,hybridization and wash compositions, and desired T_(m), those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a T_(m) of less than 45° C.(aqueous solution) or 32° C. (formamide solution), it is preferred toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found (1997)Ausubel et al, Short Protocols in Molecular Biology, page 2-40, ThirdEdit. (1997) and Sambrook et al. (1989).

Thus, isolated sequences that have regulatory element activity and whichhybridize under stringent conditions to the promoter sequences disclosedherein, or to fragments thereof, are encompassed by the presentinvention.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity” and (d)“percentage of sequence identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length promoter sequence, or the complete promoter sequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to accurately reflect thesimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm. Preferred,non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988), the local homology algorithm of Smith andWaterman (1981), the homology alignment algorithm of Needleman andWunsch (1970), the search-for-similarity-method of Pearson and Lipman(1988) and the algorithm of Karlin and Altschul (1990), modified as inKarlin and Altschul (1993).

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.,USA); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Version 8(available from Genetics Computer Group (GCG), 575 Science Drive,Madison, Wis., USA). Alignments using these programs can be performedusing the default parameters. The CLUSTAL program is well described byHiggins and Sharp (1988), Higgins and Sharp (1989), Corpet (1988), Huanget al. (1992) and Pearson (1994). The ALIGN program is based on thealgorithm of Myers and Miller (1988). The BLAST programs of Altschul etal. (1990) are based on the algorithm of Karlin and Altschul (1990). Toobtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST2.0) can be utilized as described in Altschul et al. (1997).Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules,see Altschul et al. (1997). When utilizing BLAST, Gapped BLAST orPSI-BLAST, the default parameters of the respective programs (e.g.BLASTN for nucleotide sequences, BLASTX for proteins) can be used, seethe World Wide Web site ncbi.nlm.nih.gov. Alignment may also beperformed manually by inspection.

For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity to the promotersequences disclosed herein is preferably made using the BlastN program(version 1.4.7 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an identicalor similar alignment of nucleotide matches and percent sequence identitywhen compared to the corresponding alignment generated by the preferredprogram.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid sequences makes reference to the residues in the twosequences that are the same when aligned for maximum correspondence overa specified comparison window.

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison, and multiplyingthe result by 100 to yield the percentage of sequence identity.

Identity to the sequence of the present invention would mean apolynucleotide sequence having at least 65% sequence identity, morepreferably at least 70% sequence identity, more preferably at least 75%sequence identity, more preferably at least 80% identity, morepreferably at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% sequence identity.

In accordance with one embodiment, a novel promoter is constructed bythe following steps. The sequence of a known or newly discoveredpromoter is compared with known nucleic acid sequences, such assequences in genomic databases. In one embodiment, this comparison ismade in the GenBank database using a program such as FASTA (GeneticsComputer Group, Madison, Wis.). Additional suitable databases andcomparison programs are known to a person of skill in the art. Segmentsof sequence similar to the query sequence, i.e., the known or newlydiscovered promoter, are identified and selected. Segments areconsidered similar if they have between 60% and 100% sequence identityover the segment being examined. These segments can be 20-100 bases inlength, although smaller or longer segments can also be selected. Theselected sequences are aligned in linear order according to the sequenceof the promoter being modified. The resultant promoter is a hybridpromoter comprised of sequences similar to but different from theoriginal promoter. The short segments that make up the synthetic hybridpromoter may be parts of promoters or regulatory regions from othergenes. The synthetic hybrid promoter is then constructed and empiricallytested in a test expression system to determine its quantitative andqualitative characteristics. If the synthetic hybrid promoter hasmaintained or improved activity, it may be used directly. If thesynthetic hybrid promoter has a lower activity, the sequence of thesynthetic hybrid promoter is further modified by replacing some of thebases to generate a new hybrid promoter. The new hybrid promoter isagain constructed and tested to determine if it has the desiredmaintained or improved activity. This procedure can be performed asoften as necessary to derive the final hybrid promoter having thedesired activity.

The invention is further to “functional variants” of the regulatorysequence disclosed. Functional variants include, for example, regulatorysequences of the invention having one or more nucleotide substitutions,deletions or insertions and wherein the variant retains promoteractivity, particularly the ability to drive expression preferentially tothe embryo of a plant. Functional variants can be created by any of anumber of methods available to one skilled in the art, such as bysite-directed mutagenesis, induced mutation, identified as allelicvariants, cleaving through use of restriction enzymes, or the like.Activity can likewise be measured by any variety of techniques,including measurement of reporter activity as is described at U.S. Pat.No. 6,844,484, Northern blot analysis, or similar techniques. The '484patent describes the identification of functional variants of differentpromoters.

The invention further encompasses a “functional fragment,” that is, aregulatory sequence fragment formed by one or more deletions from alarger regulatory element. For example, the 5′ portion of a promoter upto the TATA box near the transcription start site can be deleted withoutabolishing promoter activity, as described by Opsahl-Sorteberg, H-G. etal., 2004. Such fragments should retain promoter activity, particularlythe ability to drive expression of operably linked nucleotide sequences.Activity can be measured by Northern blot analysis, reporter activitymeasurements when using transcriptional fusions, and the like. See, forexample, Sambrook et al. (1989). Functional fragments can be obtained byuse of restriction enzymes to cleave the naturally occurring regulatoryelement nucleotide sequences disclosed herein; by synthesizing anucleotide sequence from the naturally occurring DNA sequence; or can beobtained through the use of PCR technology See particularly, Mullis etal. (1987) and Erlich, ed. (1989).

For example, a routine way to remove part of a DNA sequence is to use anexonuclease in combination with DNA amplification to produceunidirectional nested deletions of double stranded DNA clones. Acommercial kit for this purpose is sold under the trade name Exo-Size™(New England Biolabs, Beverly, Mass.). Briefly, this procedure entailsincubating exonuclease III with DNA to progressively remove nucleotidesin the 3′ to 5′ direction at 5′ overhangs, blunt ends or nicks in theDNA template. However, exonuclease III is unable to remove nucleotidesat 3′,4-base overhangs. Timed digests of a clone with this enzymeproduces unidirectional nested deletions.

By “promoter” is meant a regulatory element of DNA capable of regulatingthe transcription of a sequence linked thereto. It usually comprises aTATA box capable of directing RNA polymerase II to initiate RNAsynthesis at the appropriate transcription initiation site for aparticular coding sequence. The promoter is the minimal sequencesufficient to direct transcription in a desired manner. The term“regulatory element” is also used to refer to the sequence capable of“regulatory element activity,” that is, initiating transcription in adesired manner. Therefore the invention is directed to the regulatoryelement described herein including those sequences which hybridize tosame and have identity to same, as indicated, and fragments and variantsof same which have regulatory activity.

The promoter of the invention may also be used in conjunction withanother promoter. In one embodiment, the plant selection marker and thenucleotide sequence of interest can be both functionally linked to thesame promoter. In another embodiment, the plant selection marker and thenucleotide sequence of interest can be functionally linked to differentpromoters. In yet third and fourth embodiments, the expression vectorcan contain two or more nucleotide sequences of interest that can belinked to the same promoter or different promoters. For example, thepromoter described here can be used to drive the gene of interest andthe selectable marker, or a different promoter used for one or theother. These other promoter elements can be those that are constitutiveor sufficient to render promoter-dependent gene expression controllableas being cell-type specific, tissue-specific or time or developmentalstage specific, or being inducible by external signals or agents. Suchelements may be located in the 5′ or 3′ regions of the gene. Althoughthe additional promoter may be the endogenous promoter of a structuralgene of interest, the promoter can also be a foreign regulatorysequence. Promoter elements employed to control expression of productproteins and the selection gene can be any plant-compatible promoters.These can be plant gene promoters, such as, for example, the ubiquitinpromoter (European patent application no. 0 342 926); the promoter forthe small subunit of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO)(Coruzzi et al., 1984; Broglie et al., 1984); or promoters from thetumor-inducing plasmids from Agrobacterium tumefaciens, such as thenopaline synthase, octopine synthase and mannopine synthase promoters(Velten and Schell, 1985) that have plant activity; or viral promoterssuch as the cauliflower mosaic virus (CaMV) 19S and 35S promoters(Guilley et al., 1982; Odell et al., 1985), the figwort mosaic virus FLtpromoter (Maiti et al., 1997) or the coat protein promoter of TMV(Grdzelishvili et al., 2000).

The range of available plant compatible promoters includes tissuespecific and inducible promoters. An inducible regulatory element is onethat is capable of directly or indirectly activating transcription ofone or more DNA sequences or genes in response to an inducer. In theabsence of an inducer the DNA sequences or genes will not betranscribed. Typically the protein factor that binds specifically to aninducible regulatory element to activate transcription is present in aninactive form which is then directly or indirectly converted to theactive form by the inducer. The inducer can be a chemical agent such asa protein, metabolite, growth regulator, herbicide or phenolic compoundor a physiological stress imposed directly by heat, cold, salt, or toxicelements or indirectly through the actin of a pathogen or disease agentsuch as a virus. A plant cell containing an inducible regulatory elementmay be exposed to an inducer by externally applying the inducer to thecell or plant such as by spraying, watering, heating or similar methods.Any inducible promoter can be used in the instant invention. See Ward etal. (1993). Exemplary inducible promoters include ecdysone receptorpromoters, U.S. Pat. No. 6,504,082; promoters from the ACE1 system whichresponds to copper (Mett et al. (1993)); In2-1 and In2-2 gene from maizewhich respond to benzenesulfonamide herbicide safeners (U.S. Pat. No.5,364,780); the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides; andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) and McNellis et al. (1998)) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz et al. (1991), and U.S. Pat. Nos. 5,814,618 and5,789,156). Alternatively, plant promoters such as heat shock promotersfor example soybean hsp 17.5-E (Gurley et al., 1986); orethanol-inducible promoters (Caddick et al., 1998) may be used. SeeInternational Patent Application No. WO 91/19806 for a review ofillustrative plant promoters suitably employed in the present invention.

Tissue-preferred promoters can be utilized to target enhancedtranscription and/or expression within a particular plant tissue.Promoters may express in the tissue of interest, along with expressionin other plant tissue, may express strongly in the tissue of interestand to a much lesser degree than other tissue, or may express highlypreferably in the tissue of interest. Tissue-preferred promotersinclude, for example, those described in Yamamoto et al. (1997);Kawamata et al. (1997); Hansen et al. (1997); Russell et al. (1997);Rinehart et al. (1996); Van Camp et al. (1996); Canevascini et al.(1996); Yamamoto et al. (1994); Lam (1994); Orozco et al. (1993); andMatsuoka et al. (1993).

A promoter can additionally comprise other recognition sequencesgenerally positioned upstream or 5′ to the TATA box, referred to asupstream promoter elements, which influence the transcription initiationrate. Using the promoter sequences disclosed here, it is possible toisolate and identify further regulatory elements in the 5′ regionupstream from the particular promoter region identified. Thus thepromoter region disclosed is generally further defined by comprisingupstream regulatory elements such as those responsible for high leveland temporal expression of the coding sequence, enhancers and the like.In the same manner, the promoter elements which enable low to high levelexpression can be identified, isolated, and used with other corepromoters to confirm embryo-preferred expression. By core promoter ismeant the sequence sometimes referred to as the TATA box (or similarsequence) which is common to promoters in most genes encoding proteins.Thus the upstream promoter of MuB promoter can optionally be used inconjunction with its own or core promoters from other sources.

The promoter of the invention may be combined with any number of othercomponents to be introduced into the plant, including combined with anucleotide sequence of interest to be expressed in the plant. The“nucleotide sequence of interest” refers to a nucleotide sequence thatencodes for a desired polypeptide or protein but also may refer tonucleotide sequences that do not constitute an entire gene, and which donot necessarily encode a polypeptide or protein. For example, when usedin a homologous recombination process, the promoter may be placed in aconstruct with a sequence that targets an area of the chromosome in theplant but may not encode a protein. Use of antisense versions of anucleic acid sequence is another example where use of a sequence may notresult in an encoded protein. If desired, the nucleotide sequence ofinterest can be optimized for plant translation by optimizing the codonsused for plants and the sequence around the translational start site forplants. Sequences resulting in potential mRNA instability can also beavoided.

In general, the methods available for construction of recombinant genes,optionally comprising various modifications for improved expression, candiffer in detail. However, conventionally employed methods include PCRamplification, or the designing and synthesis of overlapping,complementary synthetic oligonucleotides, which are annealed and ligatedtogether to yield a gene with convenient restriction sites for cloning,or subcloning from another already cloned source, or cloning from alibrary. The methods involved are standard methods for a molecularbiologist (Sambrook et al., 1989). An expression vector is a DNAmolecule comprising a gene or antisense DNA that is expressed in a hostcell. Typically, gene expression is placed under the control of certainregulatory elements, including constitutive or inducible promoters,tissue-specific regulatory elements, and enhancers.

One skilled in the art readily appreciates that the promoter can be usedwith any of a variety of nucleotide sequences comprising the nucleotidesequence of interest to be expressed in plants. In referring to anoperably linked nucleotide sequence is intended a functional linkagebetween a promoter and another sequence where the promoter initiates andmediates transcription of the nucleotide sequence. For example, thenucleotide sequence of interest may encode a protein that is useful forindustrial or pharmaceutical purposes or the like, or to impact theplant itself, such as through expression of a protein that providesdisease resistance, insect resistance, herbicide resistance, or impactsagronomic traits as well as grain quality traits. DNA sequences nativeto plants as well as non-native DNA sequences can be transformed intoplants and used to modulate levels of native or non-native proteins. Oneor more of such sequences and/or expression cassettes may be transformedinto a plant cell (in referring to a plant cell, it is intended toinclude cells without plant membranes, such as protoplasts).

Such nucleotide sequences include, but are not limited to, thoseexamples provided below:

1. Genes that Confer Resistance to Pests or Disease

(A) Plant Disease Resistance Genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. Examples of such genes include, the tomato Cf-9 genefor resistance to Cladosporium fulvum (Jones et al., 1994), tomato Ptogene, which encodes a protein kinase, for resistance to Pseudomonassyringae pv. tomato (Martin et al., 1993), and Arabidopsis RSSP2 genefor resistance to Pseudomonas syringae (Mindrinos et al., 1994).

(B). A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon, such as, a nucleotide sequence ofa Bt δ-endotoxin gene (Geiser et al., 1986). Moreover, DNA moleculesencoding δ-endotoxin genes can be purchased from American Type CultureCollection (Rockville, Md.), under ATCC accession numbers. 40098, 67136,31995 and 31998.

(C) A lectin, such as, nucleotide sequences of several Clivia miniatamannose-binding lectin genes (Van Damme et al., 1994).

(D) A vitamin binding protein, such as avidin and avidin homologs whichare useful as larvicides against insect pests. See U.S. Pat. No.5,659,026.

(E) An enzyme inhibitor, e.g., a protease inhibitor or an amylaseinhibitor. Examples of such genes include, a rice cysteine proteinaseinhibitor (Abe et al., 1987), a tobacco proteinase inhibitor I (Huub etal., 1993), and an α-amylase inhibitor Sumitani et al., 1993).

(F) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof, such as, baculovirus expression of clonedjuvenile hormone esterase, an inactivator of juvenile hormone (Hammocket al., 1990).

(G) An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. Examples of such genesinclude, an insect diuretic hormone receptor (Regan, 1994), anallostatin identified in Diploptera punctata (Pratt, 1989),insect-specific, paralytic neurotoxins (U.S. Pat. No. 5,266,361).

(H) An insect-specific venom produced in nature by a snake, a wasp,etc., such as, a scorpion insectotoxic peptide (Pang, 1992).

(I) An enzyme responsible for a hyperaccumulation of monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(J) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, anuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. Examples ofsuch genes include, a callas gene (PCT published applicationWO93/02197), chitinase-encoding sequences (which can be obtained, forexample, from the ATCC under accession numbers 3999637 and 67152),tobacco hookworm chitinase (Kramer et al., 1993) and parsley ubi4-2polyubiquitin gene (Kawalleck et al., 1993).

(K) A molecule that stimulates signal transduction. Examples of suchmolecules include, nucleotide sequences for mung bean calmodulin cDNAclones (Botella et al., 1994) and a nucleotide sequence of a maizecalmodulin cDNA clone (Griess et al., 1994).

(L) A hydrophobic moment peptide. See U.S. Pat. Nos. 5,659,026 and5,607,914, the latter teaches synthetic antimicrobial peptides thatconfer disease resistance.

(M) A membrane permease, a channel former or a channel blocker, such as,a cecropin-β lytic peptide analog (Jaynes et al., 1993) which renderstransgenic tobacco plants resistant to Pseudomonas solanacearum.

(N) A viral-invasive protein or a complex toxin derived there from. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. See,for example, Beachy et al. (1990).

(O) An insect-specific antibody or an immunotoxin derived there from.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactive an affected enzyme, killing the insect. Forexample, Taylor et al. (1994) shows enzymatic inactivation in transgenictobacco via production of single-chain antibody fragments.

(P) A virus-specific antibody. See, for example, Tavladoraki et al.(1993), which shows that transgenic plants expressing recombinantantibody genes are protected from virus attack.

(Q) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo α-1,4-D polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase (Lamb et al., 1992). The cloningand characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart et al.(1992).

(R) A developmental-arrestive protein produced in nature by a plant,such as, the barley ribosome-inactivating gene has an increasedresistance to fungal disease (Longemann et al., 1992).

2. Genes that Confer Resistance to a Herbicide

(A) A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS (Lee et al., 1988) and AHAS enzyme (Miki et al., 1990).

(B) Glyphosate (resistance imparted by mutant EPSP synthase and aroAgenes, respectively) and other phosphono compounds such as glufosinate(PAT and bar genes), and pyridinoxy or phenoxy proprionic acids andcyclohexones (ACCase inhibitor encoding genes). See, for example, U.S.Pat. No. 4,940,835, which discloses the nucleotide sequence of a form ofEPSP which can confer glyphosate resistance. A DNA molecule encoding amutant aroA gene can be obtained under ATCC accession number 39256, andthe nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061. European patent application No. 0 333 033 and U.S. Pat. No.4,975,374 disclose nucleotide sequences of glutamine synthetase geneswhich confer resistance to herbicides such as L-phosphinothricin. Thenucleotide sequence of a phosphinothricinacetyl-transferase gene isprovided in European application No. 0 242 246. De Greef et al. (1989)describes the production of transgenic plants that express chimeric bargenes coding for phosphinothricin acetyl transferase activity. Exemplaryof genes conferring resistance to phenoxy proprionic acids andcyclohaexones, such as sethoxydim and haloxyfop, are the Accl-S1,Accl-S2 and Accl-S3 genes described by Marshall et al. (1992).

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.(1991) describes the use of plasmids encoding mutant psbA genes totransform Chlamydomonas. Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648, and DNA molecules containing thesegenes are available under ATCC accession numbers 53435, 67441 and 67442.Cloning and expression of DNA coding for a glutathione S-transferase isdescribed by Hayes et al. (1992).

3. Genes that Confer or Contribute to a Value-Added Trait

(A) Modified fatty acid metabolism, for example, by transforming maizeor Brassica with an antisense gene or stearoyl-ACP desaturase toincrease stearic acid content of the plant (Knultzon et al., 1992).

(B) Decreased phytate content

(1) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant, such asthe Aspergillus niger phytase gene (Hartingsveldt et al., 1993).

(2) A gene could be introduced that reduces phytate content. In maize,this, for example, could be accomplished by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid (Raboy etal., 1990).

(C) Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. Examples of such enzymes include,Streptococcus mucus fructosyltransferase gene (Shiroza et al., 1988),Bacillus subtilis levansucrase gene (Steinmetz et al., 1985), Bacilluslicheniformis α-amylase (Pen et al., 1992), tomato invertase genes(Elliot et al., 1993), barley amylase gene (Sogaard et al., 1993), andmaize endosperm starch branching enzyme II (Fisher et al., 1993).

The nucleotide sequence of interest can also be a nucleotide sequenceused to target an area of the plant genome through homologousrecombination. The promoter may be placed in a construct with suchsequence, which sequence will not necessarily encode a protein. Thesequence recombines in the genome and the promoter may be placed at thedesired site targeted by the sequences to regulate the desiredendogenous nucleotide sequence.

Further, the promoter can be used to drive mRNA that can be used for asilencing system, such as antisense, and in that instance, no protein isproduced. Nellen et al. (1993); Alexander et al. (1988). Means ofincreasing or inhibiting a protein are well known to one skilled in theart and, by way of example, may include, transgenic expression,antisense suppression, use of hairpin formations, co-suppression methodsincluding but not limited to: RNA interference, gene activation orsuppression using transcription factors and/or repressors, mutagenesisincluding transposon tagging, directed and site-specific mutagenesis,chromosome engineering and, homologous recombination. In the case of usewith homologous recombination, no in vivo construct will be required. Afew of the myriad of examples of such systems available include use ofthe Mu transposon, Chandler et al. (1994); RNA interference (U.S. Pat.No. 5,034,323); use of hairpins, Smith et al. (2000) and ribozymes(Steinecke et al. (1992); and zinc-finger targeted molecules, WO01/52620. Clearly many options are available for impacting a targetedprotein.

A terminator region may also be included in the vector. Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase (MacDonald et al., 1991) and nopalinesynthase termination regions. Examples of various other terminatorsinclude the pin II terminator from the protease inhibitor II gene frompotato (An et al., 1989). See also, Guerineau et al. (1991); Proudfoot(1991); Sanfacon et al. (1991); Mogen et al. (1990); Munroe et al.(1990); Ballas et al. (1989); and Joshi et al. (1987).

In one embodiment, the expression vector also contains a nucleotidesequence encoding a selectable or scoreable marker that is operably orfunctionally linked to a promoter that controls transcriptioninitiation, which can be the promoter of the invention or anotherpromoter. For a general description of plant expression vectors andreporter genes, see Gruber et al. (1993). For example, the selectivegene is a glufosinate-resistance encoding DNA or phosphinothricin acetyltransferase (pat) or a maize optimized pat gene or bar gene can be usedunder the control of the CaMV 35S or other promoter. Such pat or bargenes confer resistance to the herbicide bialaphos (Gordon-Kamm et al.,1990; Wohllenben et al. 1988). Other examples, without intending to belimiting, are hygromycin phosphotransferase, EPSP synthase anddihydropteroate encoding genes. See Miki et al. (1993). Scorable orscreenable markers may also be employed, where presence of the sequenceproduces a visual and/or measurable product. Examples include aβ-glucuronidase, or uidA gene (GUS), which encodes an enzyme for whichvarious chromogenic substrates are known (for example, U.S. Pat. Nos.5,268,463 and 5,599,670)

The expression vector can optionally also contain a signal sequencelocated between the promoter and the gene of interest and/or after thegene of interest. A signal sequence is a nucleotide sequence, translatedto give an amino acid sequence, which is used by a cell to direct theprotein or polypeptide of interest to be placed in a particular placewithin or outside the eukaryotic cell. One example of a plant signalsequence is the barley α-amylase secretion signal (Rogers, 1985). Manysignal sequences are known in the art. See, for example Becker et al.(1992), Fontes et al. (1991), Matsuoka and Nakamura (1991), Gould et al.(1989), Creissen et al. (1992), Kalderon et al. (1984) and Stiefel etal. (1990).

Leader sequences can be included to enhance translation. Variousavailable leader sequences may be substituted or added. Translationleaders are known in the art and include, for example: picornavirusleaders, for example, EMCV leader (encephalomyocarditis 5′ noncodingregion) (Elroy-Stein et al. (1989); potyvirus leaders, for example, TEVleader (Tobacco Etch Virus) (Gallie et al. (1995)); human immunoglobulinheavy-chain binding protein (BiP) (Macejak et al. (1991)); untranslatedleader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4)(Jobling et al. (1987)); tobacco mosaic virus leader (TMV) (Gallie.(1987)); and maize chlorotic mottle virus leader (MCMV) (Lommel et al.(1991)). See also, Della-Cioppa et al. (1987). Other methods known toenhance translation can also be utilized, for example, introns, and thelike. Obviously, many variations on the promoters, selectable markers,signal sequences, leader sequences, termination sequences, introns,enhancers and other components of the vector are available to oneskilled in the art.

Where appropriate, the nucleotide sequence (s) may be optimized forincreased expression in the transformed plant. That is, the genes can besynthesized using plant-preferred codons for improved expression. See,for example, Campbell and Gowri (1990) for a discussion ofhost-preferred codon usage. Methods are available in the art forsynthesizing plant-preferred genes. See, for example, U.S. Pat. Nos.5,380,831, 5,436,391, and Murray et al. (1989). Additional sequencemodifications are known to enhance gene expression in a plant. Theseinclude elimination of sequences encoding spurious polyadenylationsignals, exon-intron splice site signals, transposon-like repeats, andother such well-characterized sequences that may be deleterious to geneexpression. The G-C content of the sequence may be adjusted to levelsaverage for a given cellular host, as calculated by reference to knowngenes expressed in the host cell. When possible, the sequence ismodified to avoid predicted hairpin secondary mRNA structures.

In preparing the nucleotide construct, the various nucleotide sequencefragments can be manipulated, so as to provide for the nucleotidesequences in the proper orientation and, as appropriate, in the properreading frame. Toward this end, adapters or linkers can be employed tojoin the nucleotide sequence fragments or other manipulations may beinvolved to provide for convenient restriction sites, removal ofsuperfluous nucleotide sequences, removal of restriction sites, or thelike. For this purpose, in vitro mutagenesis, primer repair,restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Methods for introducing expression vectors into plant tissue availableto one skilled in the art are varied and will depend on the plantselected. Procedures for transforming a wide variety of plant speciesare well known and described throughout the literature. See, forexample, Miki and McHugh (2004); Klein et al. (1992); and Weising et al.(1988). For example, the DNA construct may be introduced into thegenomic DNA of the plant cell using techniques such asmicroprojectile-mediated delivery (Klein et al. 1992), electroporation(Fromm et al., 1985), polyethylene glycol (PEG) precipitation (Mathurand Koncz, 1998), direct gene transfer (WO 85/01856 and EP-A-275 069),in vitro protoplast transformation (U.S. Pat. No. 4,684,611), andmicroinjection of plant cell protoplasts or embryogenic callus(Crossway, 1985). Agrobacterium transformation methods of Ishida et al.(1996) and also described in U.S. Pat. No. 5,591,616 are yet anotheroption. Co-cultivation of plant tissue with Agrobacterium tumefaciens isa variation, where the DNA constructs are placed into a binary vectorsystem (Ishida et al., 1996). The virulence functions of theAgrobacterium tumefaciens host will direct the insertion of theconstruct into the plant cell DNA when the cell is infected by thebacteria. See, for example, Fraley et al. (1983). Agrobacterium isprimarily used in dicots, but monocots including maize can betransformed by Agrobacterium. See, for example, U.S. Pat. No. 5,550,318.In one of many variations on the method, Agrobacterium infection of corncan be used with heat shocking of immature embryos (Wilson et al. U.S.Pat. No. 6,420,630) or with antibiotic selection of Type II callus(Wilson et al., U.S. Pat. No. 6,919,494).

Rice transformation is described by Hiei et al. (1994) and Lee et al.(1991). Standard methods for transformation of canola are described byMoloney et al. (1989). Corn transformation is described by Fromm et al.(1990) and Gordon-Kamm et al. (1990). Wheat can be transformed bytechniques similar to those used for transforming corn or rice. Sorghumtransformation is described by Casas et al. (1993) and barleytransformation is described by Wan and Lemaux (1994). Soybeantransformation is described in a number of publications, including U.S.Pat. No. 5,015,580.

In one preferred method, use of aerosol beam technology for introductionof nucleotide sequences into cells is employed. Aerosol beam technologyemploys the jet expansion of an inert gas as it passes from a region ofhigher gas pressure to a region of lower gas pressure through a smallorifice. The expanding gas accelerates aerosol droplets containing themolecules to be introduced into a cell or tissue. Aerosol dropletsproduced are typically less than 0.1 micron in diameter at the point ofimpact with the target cells. DNA carried in aerosol droplets of thissmall size penetrates cells only because of the speeds attained by theaerosol droplets. Speeds achieved by the aerosol beam method of theinvention are supersonic and can reach 2000 meters/second. In apreferred embodiment, the process includes (I) culturing a source ofcells, (II) optionally, pretreating cells to yield tissue with increasedcapacity for uptake and integration by aerosol beam technology, (III)transforming said tissue with an exogenous nucleotide sequence by theaerosol beam method of the invention, (IV) optionally, identifying orselecting for transformed tissue, (V) optionally regenerating transgenicplants from the transformed cells or tissue, and (VI) optionally,producing progeny of said transgenic plants. This process is describedin detail at Held et al., U.S. Pat. Nos. 6,809,232; 7,067,716; and7,026,286 (these references, as all cited references, are incorporatedherein by reference).

In accordance with the present invention, a transgenic plant can beproduced that contains an introduced MuB promoter. It can be combinedwith any one of the components set forth above.

In a further embodiment, plant breeding can be used to introduce thenucleotide sequences into other plants once transformation has occurred.This can be accomplished by any means known in the art for breedingplants such as, for example, cross pollination of the transgenic plantsthat are described above with other plants, and selection for plantsfrom subsequent generations which contain the nucleic acid and/orexpress the amino acid sequence or trait. The plant breeding methodsused herein are well known to one skilled in the art. For a discussionof plant breeding techniques, see Poehlman and Sleper (1995). Many cropplants useful in this method are bred through techniques that takeadvantage of the plant's method of pollination. A plant isself-pollinating if pollen from one flower is transferred to the same oranother flower of the same plant. A plant is cross-pollinating if thepollen comes from a flower on a different plant. For example, inBrassica, the plant is normally self-sterile and can only becross-pollinated unless, through discovery of a mutant or throughgenetic intervention, self-compatibility is obtained. Inself-pollinating species, such as rice, oats, wheat, barley, peas,beans, soybeans, tobacco and cotton, the male and female plants areanatomically juxtaposed. During natural pollination, the malereproductive organs of a given flower pollinate the female reproductiveorgans of the same flower. Maize plants (Zea mays L.) can be bred byboth self-pollination and cross-pollination techniques. Maize has maleflowers, located on the tassel, and female flowers, located on the ear,on the same plant. It can self or cross-pollinate.

Pollination can be by any means, including but not limited to hand, windor insect pollination, or mechanical contact between the male fertileand male sterile plant. For production of hybrid seeds on a commercialscale in most plant species pollination by wind or by insects ispreferred. Stricter control of the pollination process can be achievedby using a variety of methods to make one plant pool male sterile, andthe other the male fertile pollen donor. This can be accomplished byhand detassling, cytoplasmic male sterility, or control of malesterility through a variety of methods well known to the skilledbreeder. Examples of more sophisticated male sterility systems includethose described by Brar et al., U.S. Pat. Nos. 4,654,465 and 4,727,219and Albertsen et al., U.S. Pat. Nos. 5,859,341 and 6,013,859.

Backcrossing methods may be used to introduce the gene into the plants.This technique has been used for decades to introduce traits into aplant. An example of a description of this and other plant breedingmethodologies that are well known can be found in references such asPoehlman et al. (1995). In a typical backcross protocol, the originalvariety of interest (recurrent parent) is crossed to a second variety(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a plantis obtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the single transferred gene from thenonrecurrent parent.

EXAMPLES

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized. Inthese Examples, corn is used for illustrative purposes only. Other plantspecies are also transformed with the DNA constructs of the presentinvention using techniques well known in the art.

Example 1 Construction of Plasmids Used in Corn Transient Assays

A promoter sequence, designated Arabidopsis homolog to the CaMV 35S, wasdesigned with around 80% homology to the CaMV 35S promoter (FIG. 1) andsegments with homology to sequences from Arabidopsis as shown in FIG. 2:A: MuA U.S. Pat. No. 6,222,096; B: AB000835; C: Y08502; D: U09376; E:AB005247; F: AF028711; G: AC002521; H: D12522; I: AB012243; J: AL022347.The sequence was not randomly derived but based on homologies found inthe database at the time of design. Three hundred fifty one bases of theCaMV 35S promoter were compared to plant DNA sequences, mainly fromArabidopsis, in the database GenBank using the FASTA program. Regions ofhomologies were then manually aligned using the CaMV 35S promoter as thetemplate. A few bases within segments and in regions linking segmentswere changed to match the CaMV 35S sequence so that the homology wouldremain around 80%. The comparison of the MuB promoter with the CaMV 35 Spromoter is shown in FIG. 3. Homology to the MuA promoter of U.S. Pat.No. 6,222,096 was about 70%. The comparison of the MuB promoter with theMuA promoter is shown in FIG. 4. The sequence was synthesized by theMolecular Biology Group at The Midland Certified Reagent Company,Midland, Tex. with EcoRI and SacI compatible overhangs on the 5′ and 3′ends, respectively.

Plasmid 350096 was constructed by removing a root preferential promoterwith an EcoRI and SacI double digestion from the plasmid pZMOO96 of U.S.Pat. No. 5,633,363 and replacing it with a CaMV 35S promoter frompSLJ4k1 which had compatible EcoRI/SacI cohesive ends. Plasmid pSLJ4k1was obtained from the Sainsbury Laboratory at the John Innes Center,England. Plasmid 350096 consisted of a 1.3 kb CAMV 35S promoter, 0.5 kbintervening sequence six (IV6) from corn alcohol dehydrogenase, 1.8 kbGUS reporter gene, 0.25 kb nopaline syntase 3′ end (nos) in a pUC18backbone.

The CaMV 35S promoter in p350096 was replaced with the synthesizedArabidopsis homolog. The plasmid p350096 was digested with EcoRI andSacI, then dephosphorylated to prevent self ligation. The homolog wasdirectionally ligated into the EcoRI and SacI sites to produce pY0096.

Example 2 Transient Assays in Corn Callus

Tissue preparation: Type II callus of Stine elite inbred 963 was used astarget tissue for transient assays. The Type II callus, initiated fromimmature embryos on N6AMOD medium (Table 1), was maintained as a stockculture on DN62 medium (Table 1). Tissue was transferred every ten days.For transient assays tissue was taken from these cultures (usually 4 to5 days after subculture) and spread on a Whatman No 4 filter disk onDN620SM medium (Table 1) up to one day prior to bombardment with theparticle inflow gun.

DNA Delivery: A particle inflow gun (PIG) as described by Finer et al.(1992) and Vain et al. (1993) was used to deliver the DNA. In brief, 50mg of tungsten particles (M10 from Sylvania Chemicals/Metals, Towanda,Pa.) were sterilized for 15 minutes in 95% ethanol in a 1.5 ml microfugetube. Particles were rinsed 3 times in sterile distilled water byrepeated vortexing, centrifugation and resuspension in 0.5 ml water.Particle suspensions were made fresh for each experiment. Plasmid DNAwas coated onto the particles by mixing 25 ul of tungsten particlesuspension (2.5 mg), 5 μl of DNA (5 ug), 25 ul of 2.5 M CaCl.sub.2, and10 ul of 100 mM spermidine (free base). After allowing the particles tosettle for a few minutes while on ice, 50 ul of supernatant was removed.Two ul of the remaining particle suspension was pipetted onto the centerof the screen of a syringe filter unit. The syringe filter unit wasreassembled and screwed into the Luer-lok needle adaptor within thechamber. The target tissue in a petri plate was placed about 15 cm belowthe syringe filter unit. A vacuum of approximately 28 in Hg was appliedand the particles were discharged when helium (80 psi) was releasedfollowing activation of the solenoid by the timer relay.

TABLE 1 Media Compositions Ingredients/L N6AMOD DN62 DN62OSM N6 salts¹3.98 g 3.98 g 3.98 g N6 vitamins² 1 ml 1 ml 1 ml Asparagine 150 mg 800mg 800 mg Myo-inositol 100 mg 100 mg 100 mg Proline 700 mg 1400 mg 1400mg Casamino Acids 100 mg 100 mg 2,4-D 1 mg 1 mg 1 mg Sucrose 20 g 20 g20 g Glucose 10 g MES 500 mg Mannitol 45.5 g Sorbitol 45.5 g AgNO₃ 10 mgGelrite 2 g 3 g 3 g pH 6.0 5.8 5.8 ¹N6 salts - Sigma Plant CultureCatalogue ref. C 1416. ²N6 vitamins: 2 mg/l glycine, 0.5 mg/l nicotinicacid, 0.5 mg/l pyridoxine HCl, 1 mg/l thiamine HCl (Chu, 1978).

After bombardment, callus was incubated at 25° C. in the dark for 16-24hours on the same medium used for bombardment. Then, transient GUSexpression was evaluated by incubating the tissue in 0.5 mg/ml X-gluc(Gold Biotechnology, Inc. St. Louis, Mo.) in 0.1M sodium phosphatebuffer pH 7.0 and 0.1% Triton-X-100 at 37.degree. C. for 4-16 h afterwhich the number and intensity of blue foci were evaluated under astereo microscope at approximately 10× magnification. Tissue wastransformed with either p350096, or pMuB0096. Tissue transformed withp350096 or pMuB0096 had similar levels of transient expression. Tissuetransformed with pY0096 was found to have lower levels of transientexpression.

Example 3 Construction of Plasmids Used in the Transformation of Corn

The bar gene in pBARGUS (Fromm et al., 1990) was used to replace the GUSgene in pMuB0096 using the BamHI and PstI sites. Briefly, pBARGUS wasdigested with BamHI and PstI and the 800 bp fragment containing the bargene was isolated. The plasmid pMuB0096 was digested with BamHI and PstIand the ends were dephosphorylated. The 800 bp fragment containing thebar gene was ligated to the digested, dephosphorylated pMuB0096 toproduce the plasmid pMuBBar.

The MuBBar expression cassette was removed from pMuBBar by digestionwith EcoRI and HindII and cloned into pSB11 (obtained from JapanTobacco) after digestion with EcoRI and HindIII and dephosphorylation,resulting in the plasmid pSB11MuABar. Plasmid pSB11MuABar was combinedwith pSB1 (obtained from Japan Tobacco) via homologous recombination andmobilized into Agrobacterium LBA 4404 via triparental mating accordingto U.S. Pat. No. 5,591,616 resulting in pSBMuBBar. This plasmid in LBA4404 was used in the transformation of corn.

Example 4 Transformation of Corn

Agrobacterium-mediated DNA delivery was used to produce stable corntransformants carrying MuB driving the bar gene. Agrobacterium LBA 4404harboring PSB11MuBBar recombined with pSB 1 (Example 3) was taken fromglycerol stocks and streaked out on YP medium (5 g/l yeast extract, 10g/l peptone, 5 g/l NaCl, 15 g/l agar and pH 6.8) supplemented with 50mg/l spectinomycin and grown for one or two days at 28.degree. C.

Immature embryos of Stine elite inbred 963 were aseptically removed fromkernels of plants grown in a grow room (15 h photoperiod, 28° day and25° night). Embryos were harvested 10 to 11 days after pollination whenthey were between 1 mm and 2 mm in length and then placed in 2 ml ofLSinf medium (Table 2) in an Eppendorf tube. The mixture was thenstirred with a vortex mixer (Vortex Genie 2) at full speed for 5seconds, the LSinf removed, replaced with fresh medium and then stirredagain. All medium was then removed from the tube using a Pasteurpipette. Bacteria were collected with a platinum loop (enough to coatthe wire of the loop) and thoroughly suspended in 1 ml of Lsinf-ASmedium (Table 2) using a Pasteur pipette. The bacterial suspension wasthen introduced into the tube containing the embryos and the mixturestirred with a vortex mixer at full speed for 30 seconds. After this theembryos were allowed to stand for five minutes and were then transferredto the surface of LSAS medium (Table 2) solidified with agar, care beingtaken to remove any accompanying liquid. Embryos were immediatelyoriented so that the scutellar surface was uppermost.

TABLE 2 Media Compositions Ingredients/L LSAS Lsinf LsinfAS MSsalts/vits¹ 4.43 g 4.43 g 4.43 g Proline 700 mg Casamino Acids 1.0 g 1.0g Na₂ EDTA 37.3 mg 37.3 mg 37.3 mg 2,4-D 1.5 mg 1.5 mg 1.5 mg MES 500 mgThiamine HCl 1.0 mg 1.0 mg Sucrose 20 g 68.5 g 68.5 g Glucose 10 g 36.0g 36.0 g Acetosyringone 100 μm 100 μm Phytagar 7 g pH 5.8 5.2 5.2 ¹MSsalts/vits - Sigma Plant Culture Catalogue ref. M5519

The embryos were then cultured in the dark at 19° for 48 hours. Afterthis time the plates were removed from the incubator and placed at 45°for 30 minutes. Then they were returned to the 19° incubator for afurther day. Following this the embryos were transferred to DN62ALC(Table 3) and incubated at 24° for 5 days. Next, the embryos weretransferred to DN62ALCB (Table 3) and incubated at 24° for 14 days. Forthe next 14-day passage the cefotaxime concentration was raised from 50mg/l (in DN62ALC) to 250 mg/l. This medium—DN62ACB (Table 3)—allowed fora better control of the residual Agrobacterium cells contaminating thecorn embryos. Embryos were then transferred back to DN62ALCB for afurther 14 days. At this time, transformed corn clones could berecognized by their ability to grow as prolific Type II callus on thebialaphos-containing medium. Culture of the clones continued on DN62Bmedium (see Table 3) for a further two weeks after which time the TypeII callus was transferred to DNROB medium (Table 4) to initiateregeneration. After one to two weeks on DNROB somatic embryos developedas individual structures. These embryos were allowed to mature for oneto two weeks on a further passage of DNROB (Table 4) and were thentransferred to DNO6S (Table 4). Finally, they were transferred to MSOGor ½MS0.1IBA (Table 4) where they germinated and formed plantlets. Theplantlets were then transferred to tubes containing ½MS 0.1IBA whereroots developed. The plants were transferred to peat pots prior to goinginto the greenhouse. In the greenhouse the plants were grown to maturityand seed collected either after backcrossing to Stine inbred 963 orafter selfing.

The presence of an expressing bar gene was then confirmed by leafpainting with the agricultural herbicide, LIBERTY, both in the primarytransformants and in progeny. Mendelian ratios of an expressing bar genewere routinely observed in the progeny.

TABLE 3 Media Compositions Ingredients/L DN62B DN62ALC DN62ALCB DN62ACBN6 salts¹ 3.98 g 3.98 g 3.98 g 3.98 g N6 vitamins¹ 1 ml 1 ml 1 ml 1 mlAsparagine 800 mg 800 mg 800 mg 800 mg Myo-inositol 100 mg 100 mg 100 mg100 mg Proline 1400 mg 1400 mg 1400 mg 1400 mg Casamino Acids 100 mg 100mg 100 mg 100 mg 2,4-D 1 mg 1 mg 1 mg 1 mg Sucrose 20 g 20 g 20 g 20 gGlucose 10 g AgNO₃ 10 mg 10 mg 10 mg Bialaphos 1 mg 1 mg 1 mg Cefotaxime50 mg 50 mg 250 mg Gelrite 3 g 3 g 3 g 3 g pH 5.8 5.8 5.8 5.8 ¹N6 saltsand vitamins as in Table 1.

TABLE 4 Media Compositions Ingredients/L DNROB DNO6S MSOG ½ MS 0.1IBA MSSalts 4.43 g 4.43 g 4.43 g 2.215 g Asparagine 800 mg Proline 1400 mg Na₂EDTA 37.3 mg 37.3 mg 37.3 mg 7.3 mg Casamino Acids 100 mg Nicotinic Acid0.5 mg Gibberellic Acid 0.1 mg Indole-3-Butyric 0.1 mg Acid Sucrose 60 g30 g 20 g Sorbitol 20 g Bialaphos 1 mg Gelrite 2 g 2 g Phytagar 7 g 7 gpH 5.8 5.8 5.8 5.8 ¹MS Salts - Sigma Plant culture Catalogue ref. M5519

Example 5 Transformation of Soybean

The plasmid pMuBGUS35N was transformed into soybean using aerosol beamtechnology as described in U.S. Pat. No. 6,809,232. The plasmidMuBGUS35N contained two expression cassettes: 1) MuB promoter linked tothe GUS gene with a NOS terminator and 2) 35S promoter linked to anNPTII gene with an OCS terminator. The NPTII expression cassettefacilitated the selection of transformed callus on media supplementedwith 300 mg/l kanamycin.

Embryogenic soybean callus was transferred after a culture passage ofabout 28 to 30 days from stock culture medium (B1-30 3Co5My 50 mg/lphytic acid—Table 5) to the center of a target plate containing the samemedium. Embryogenic soybean callus can survive being held in a vacuumfor at least 10 minutes. After one to three days' growth on the targetplate, the soybean embryogenic callus is exposed to an aerosol beam ofpMuBGUS35N (the MuB promoter driving the GUS gene). After beaming thetissue is spread out on a fresh plate (to minimize the risk ofcontamination) of the same medium.

TABLE 5 Growth Media for Soybean* Ingredients in 1 Liter B1-30 B3 B5G MsSalts 4.43 g 4.43 g B5 Salts 3.19 g NaEDTA 37.3 mg 37.3 mg 37.3 mg 2,4-D30 mg Activated 5 g Charcoal Phytagar 8 g 8 g Gelrite 2 g pH 5.8 5.8 5.8*Variations of media referred to in Table 5 were tested, e.g., B1-303Co5My, which was made by adding 3% coconut water and 5 gm/l myoinositolto B1-30. Other variations included: B1-30 3Co5My0.25 PA0.5K whichcontained B1-30 basal medium plus 3% coconut water, 5 gm/l myoinositol,0.25 gm/l phytic acid, and 0.5 gm/l additional KH₂PO₄ and ½ B5G whichcontained all ingredients of B5G medium at half strength.

Briefly, the treatment of target tissue with the aerosol beam apparatuswas performed as follows: 1) place petri dish with tissue on the stageand close vacuum chamber; 2) start the vacuum pump; 3) start the syringepump; 4) set the nebulizing gas pressure; 5) set the entrainment gaspressure, and by this time the correct vacuum in the chamber is reached;and 6) start the movement of the stage and let the system run for thetime needed to complete the run. After the run is completed, shut downthe stage, vacuum, syringe pump, nebulizing gas, entrainment gas, andremove target tissue from the chamber.

The aerosol was produced by a microflow nebulizer such as the HEN fromJ.E. Meinhard Associates Inc., or the MCN100 style M4 nebulizer fromCetac Technologies Inc. (Liu and Montaser, 1994; Tan, et al., 1992). Thenebulizing gas was high purity compressed-helium which was regulatedwith an ACCU-TROL gas regulator—876X model RS-7-4 and filtered throughan Arrow F300-02 IT filter. When HEN and the MCN100 microflow nebulizerswere used, the nebulizing pressure was preferably 20-30 psi but workedwithin the range from about 10 psi to about 40 psi. The entrainment gasfilled the entrainment tube and entrained the aerosol droplets in astraight line. Unfiltered, high purity compressed helium was used as theentrainment gas and was regulated by an Arrow R262 regulator to produceslight positive pressure as measured by a Gilmont model 65 mm gauge. Theentrainment housing contained a nucleospot to reduce electrostaticcharges and was maintained at a temperature of about 42° C. to about 55°C., and most preferably about 55° C. This reduced coalescing of theaerosol droplets and was controlled by two Omega CN9000 seriestemperature controllers. The sample flow rate to the nebulizer wascontrolled by a Harvard 11 infusion only syringe pump. The flow rate was1 to 1200 μl/min using a sterile Becton Dickinson 1 cc plastic syringewith a 0.2 micron filter attached. The sample contained 10 mM Trisbuffer (pH 7.0) or a carbohydrate molecule (for example 1 g/l sucrose)and the molecules to be delivered.

Approximately one day after treating the soybean callus with the aerosolbeam apparatus, transient expression was evaluated by histochemicalanalysis. Embryogenic callus was incubated in the presence of thesubstrate X-gluc (Gold Biotechnology, Inc.) at a concentration of 0.5mg/ml in 0.1 M sodium phosphate buffer pH 7.0 and 0.1% Triton-x-100 at37° C. After 1-4 hours blue spots appeared indicating GUS expression.

Plants were regenerated as described in U.S. Pat. No. 6,809,232 andassayed for GUS activity, as described supra. Strong GUS activity wasobserved in leaves, roots and stem.

Thus MuB is a functional promoter with the characteristics of aconstitutive promoter and useful in the genetic engineering of plants.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

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1. A isolated regulatory element comprising a nucleic acid molecule selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 1; (b) a sequence having more than 80% identity to SEQ ID NO: 1; and (c) A sequence comprising a functional fragment of at least 71 bases of the nucleotide sequence set forth in SEQ ID NO: 1, wherein the fragment exhibits regulatory element activity.
 2. An expression vector comprising a regulatory element selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 1; (b) a sequence having more than 80% identity to SEQ ID NO: 1; (c) a nucleotide sequence which hybridizes to SEQ ID NO: 1 under highly stringent conditions of a wash of 50% formamide, 1.0 M NaCl, 0.1% SDS at 37° C., and a wash in 0.1×SSC at 60° C., wherein the sequence exhibits regulatory element activity; and (d) a sequence comprising a functional fragment of at least 71 bases of the nucleotide sequence set forth in SEQ ID NO: 1, wherein the fragment exhibits regulatory activity.
 3. The expression vector of claim 2 further comprising a second nucleotide sequence operably linked to the regulatory element, the second nucleotide sequence capable of conferring a selected agronomic trait to a plant.
 4. The vector of claim 3 wherein the agronomic trait is selected from the group consisting of herbicide resistance, insect resistance, disease resistance, drought tolerance, salt tolerance and increased yield.
 5. A plant comprising the expression vector of claim
 2. 6. A plant cell comprising the expression vector of claim
 2. 7. A method of producing a transformed plant cell, the method comprising transforming a plant cell with the nucleic acid molecule of claim
 1. 